Test methods are generally means with which the functioning of human and animal organs is observed and changes perceived. Such methods involve chemical/biochemical analyses, continuous registrations and histological checks.
A special group of test methods are analysis methods, with the help of which nature and quantity of medicaments, stimulants, addictive substances and environmental poisons can be determined, e.g. blood alcohol determination, cardiac glycoside determination etc. Of the biological fluids to be investigated blood, blood plasma and urine are the most important, others including faeces, gland secretions such as saliva, gastric juice, bile, pancreatic juice, sweat, spinal fluid and exudates in the breast, stomach and joints. Examinations on organes are governed by the problem posed.
In view of the manifold requirements to be met by a diagnostic agent with regard to checking on the course of a disease, therapy checking and therapy control, and of the large amounts needed, repeated application is at first restricted to simple, inexpensive methods; with time, one will change over more and more to specific diagnostic agents for follow-up examinations.
Due to the continuous search for methods which are more convenient to handle and also more specific due to the fact that more and more examinations are carried out on the same object only once instead of several times and due to the non-toxicity of a substance category (in-vitro diagnosis) and not least for reasons of health politics, i.e. preventive medical checkups for the entire population, and the want of the individual to have preventive checkups, the importance of checking methods has been continuously increasing in recent times.
Functional diagnostic agents for checking on the physiological functioning of individual organs and organ systems play a special role among the checking methods. Related to these diagnostic agents are those that stimulate secretion by endocrine glands, thus providing a measure of the functional efficiency of, in particular, the stomach, pancreas etc.
Test methods for obtaining a functional diagnosis to assess how detrimental a substance is in respect of its cellulotoxic effect have already been studied by observing the formation of plasma polypi (PP).
In the publication Res. exp. Med. 171 (1977), pages 155 to 162, for example, experiments are described pertaining to the in-vitro production of plasma polypi (PP) found in human placentae after birth, in order to concentrate the polypi in one operation using reverse centrifugation and to obtain them as pure as possible.
In the publication "Archiv fur Gynakologie", 244 (1977), page 195 investigations are described concerning the commonness of acetylsalicylic acid in the placenta following parenteral administration to the mother during the birth, and in "Fortschr. med. 89 (1971)" pages 1159 to 1162, there is a report on the side-effects of salicylates on the human organism.
It is also already known that the poisoning of pregnant guinea pigs with mono-iodine-acetate or sodium fluoride as inhibitor of carbohydrate catabolism leads within a few minutes to significant morphological changes at the synctytiotrophoblast of the placenta. Besides swelling of the mitochondria, of the endoplasmic reticulum and of the Golgi's cisternae, as well as a nuclear pyknosis, a large number of plasma polypi occur (protrusions, 0.5 to 20 .mu.m in size, with few organelles). In long-term experiments monitored for up to 10 days, these PP were found to lead to placenta infarcts. Histological examinations of human placentae which, following incubation in a shaker bath, had been cut into small strips also showed increased PP formation from the syncytiotrophoblast.
Increased formation of plasma polypi as symptom of pregnancy disorder is known from the publication "Archiv der Gynakologie", 216 (1974), pages 175 to 176.
In experimental investigations carried out on animals to systematically research the causes of PP formation it was possible, by way of administering sodium fluoride (NaF) or mono-iodine-acetate to inhibit glucose catabolism, to obtain an increased, reproducible formation and pinching-off of cell protrusions in guinea-pig placentae.
The difficulty of assessing the results obtained by known determination methods is manifested particularly in the fact that many substances that have an effect on human or animal organisms, damage only becomes apparent after repeated application or prolonged periods of contact, e.g. softening agents in plastics, halogenated products, etc., going unnoticed to start with and then only being demonstrable through the use of complicated examination procedures. Since up to now it has not always been possible to demonstrate toxic effects simply and quickly with histological specimens, one was forced to carry out systematic series of tests on laboratory animals, which were expensive and time consuming.
The pinching-off of cell protrusions as a result of the effect of toxic substances and mechanical lesions, is a feature of all animal and human tissues, independent of the sex, e.g. tested on cell cultures of blood strain cells, hepatocytes, fibroblasts etc.
This result does not only apply to the syncytiotrophoblast, but also to anucleate erythrocytes. In the syncytiotrophoblast, which causes the exchange of blood gases of any one of substrates and the metabolic end products between mother and foetus, the cellular membrane is catabolized very quickly. Therefore, most tissues are ruled out for quantitatively registering of cell protrusions, because the PPs cannot be extracted quantitatively from the tissue in its entirety. For quantitatively registering PPs a comparitavely large and accessible tissue surface is therefore required as is present, e.g. in the case of the placenta (see DE-PS No. 28 10 425). Registration carried out on the placenta employing said known method, however, was only applicable after the placenta had been completely rinsed off of blood and by determination of the PPs by way of reversed centrifugation, which was very time-consuming and necessitated fully developed animal and human placentae. This applies also to the possibility of observing the effect of toxic substances on individual villi of human placentae, in particular with regard to the period of formation and size of the PPs.
The present invention has the object of providing a test method with which toxic or undesirable side-effects of known and of new substances can be determined more quickly and more reliably than previously, and easily demonstrated.
Subject matter of invention is accordingly a method for testing chemical and biological substances intended for or contained in medicaments, food, insecticides, pesticides, solvents and other everyday goods for their cellulotoxic effect, which method involves treating animal or human tissue with the substances in question--in combination with sodium pyruvate, if required--so as to form plasma polypi (protrusions containing few cell organelles), which serve as a measure of the cellulotoxic effect, and, the substances having been allowed time to act on the excised tissue treated, determining the content of plasma polypi, characterized in that, by taking random samples, in the excised tissue the plasma polypi (PP) are determined qualitatively and over the entire excised gastric mucosa quantitatively using histological sections.
Most medicaments, substances needed every day and food first of all enter the stomach, where they remain for varying length of time. If the administered substances contain toxic substances the length of time and form of administration is demonstrable in a well defined and reproducible form.
The present invention is accordingly based on recognition of the fact that the formation of plasma polypi is a generally useful and reliable indicator of a certain type of cellulotoxic effect attributable to the test substance in question.
The test method according to the invention is of manifold application for determining whether the substance being tested is harmful to human or animal organs. In order to test the toxic effect in relation to the dose, use is made of a sex-independent tissue, namely the gastric mucosa.
The method used in the present invention for determining the content of plasma polypi on the gastric mucosa can be carried out in every industrial company or government laboratory.
Contrary to the conventional methods used for plasma polypi determination, for example at the placenta, the pinched-off plasma polypi formed on the large surface of the gastric mucosa pass directly into the lumen of the stomach free of foreign particles, i.e. free of blood and other tissue particles and can be extracted from the lumen of the stomach by removing and preparing the entire gastric tissue using standardized cuttings at the cardia and pylorus. Such extraction of PPs which are free of foreigh particles is surprinsing and cannot be inferred from the prior art. Moreover, this method represents a great methodical adavance both with respect to its handling and the possibility of early diagnosis of damaging effects.
Contrary to the conventional test procedures, the method according to the invention represents an easy way of providing a quantitative measure of any cellulotoxic effect. The new method has the added advantage that gradual cellulotoxic effects of a substance, which up to now could only be histologically qualitatively detected after a long duration of action and delay, can now be determined with a good degree of reliability quantitatively and very quickly.
By conducting tests on the gastric mucosa it is easy to quantitatively determine the damage by counting the plasma polypi. In addition to this, the method of the invention makes it possible to determine the number of plasma polypi as a function of dose and duration of action.
It is thus possible, using simple histological sections, to determine--as a function of time--e.g. the damage done by acetyl salicylic acid also in the lower layers of cells. This allows one, in cases of repeated administration or raised individual doses, to observe all the stages of the damage, from simple lesions of the mucosa, ulceration and finally bleeding, since repeated administration or an overdose cause damage to the vascular endothelium as well.
By detecting and evaluating the PP one is able to make dose-dependent checks at intervals for possible damage or undesirable side-effects which any one substance may cause. This was up till now not possible.
Counting and determination of the size distribution are conducted microscopically, anything which is particularly unusual as regards shape or contents being photographed.
PP formation on the gastric mucosa occurs on individual cells that exhibit a relatively uniform size distribution of 1.0 to 3.0 .mu.m diameter. Quantitative information on the toxic effect of a substance can be obtained within 1 day using the method of the present invention.
In the method according to the invention guinea pigs, hamsters, rats, mice, dogs, cats, pigs and monkeys are used as experimental animals, the substance to be tested, e.g. acetyl salicylic acid, pyridoxal phosphate, pyridoxal, formaldehyde, mono-iodine-acetate or the mixture to be tested comprising toxic substance and sodium pyruvate, being administered intravenously, intraperitoneally or intramuscularly or perorally.
With regard to the cellulotoxic effect, these animals react in a similar way to humans.
For closer details of the invention, attention is drawn to the test examples.
A positive test is proof of a toxic substance with increased PP formation on the gastric mucosa. Here, it has to be contemplated, for example, withdrawing the toxic substance from the market or combining it with another substance compensating for the cellulotoxic effect of said first toxic substance. Examples of such protective substances include: Na pyruvate, Na lactate, methylglyoxale, alanine, serine, glutamic acid, glutamine, aspartic acid, asparagine, 2-oxodicarboxylic acids and 2-oxotricarboxylic acids.
PP formation in the gastric mucosa of the rat occurs primarily at the surface layers of cells of the gastric mucosa. The plasma polypi are shed directly into the lumen of the stomach.
The PPs measure 1 to 5 .mu.m, especially 1 to 2 .mu.m, in diameter. PPs measuring 3 to 5 .mu.m are the exception and account for only 3 to 5% of the total number.
Following administration of test substances such as acetyl salicylic acid (ASS) one can observe, depending on the length of time the substance remains in the stomach, when deeper layers of the gastric mucosa are affected and what effect the test substance has on vascular endothelium, i.e. whether bleeding occurs. The blood passes together with the PPs into the lumen of the stomach.